Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.
Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.
复制标题
DOI：
10.1073/pnas.93.19.10134
复制
发表时间：
1996-09
影响因子：
11.1
通讯作者：
R. Yao;Erasmus Schneider;T. J. Ryan;J. Galivan
中科院分区：
文献类型：
--
作者：
R. Yao;Erasmus Schneider;T. J. Ryan;J. Galivan
通过搜索表达序列标签数据库并使用大鼠γ-谷氨酰水解酶cDNA作为查询序列，已经鉴定了编码人γ-谷氨酰水解酶的cDNA。该cDNA编码Mr 35,960的318个氨基酸的蛋白质。推导的人γ-谷氨酰水解酶的氨基酸序列与大鼠γ-谷氨酰水解酶的氨基酸序列有67%的同一性。在大鼠和人类中，N 末端之前的 24 个氨基酸构成类似于前导序列或信号序列的结构基序。人类序列中有四个共有的天冬酰胺糖基化位点，其中三个在大鼠酶中是保守的。当甲氨蝶呤二或五谷氨酸作为底物时，人和大鼠 cDNA 在大肠杆菌中的表达分别产生具有天然人和大鼠酶特征的抗原相关蛋白。利用后一种底物，大鼠酶裂解最内部的γ-谷氨酰键，导致仅产生甲氨蝶呤作为含蝶酰基的产物。人类酶的不同之处在于，它首先产生甲氨蝶呤四谷氨酸，然后是三谷氨酸，然后是二谷氨酸和甲氨蝶呤。因此，大鼠酶是以甲氨蝶呤五谷氨酸为底物的内肽酶，而人酶表现出外肽酶活性。另一个区别是表达的大鼠酶对甲氨蝶呤二谷氨酸和五谷氨酸具有相同的活性，而人酶对甲氨蝶呤五谷氨酸的活性比二谷氨酸高几倍。这些特性与源自人类和大鼠的酶一致。
A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources.